Melanoma-Determining Tu Loci in Xiphophorus

In Xiphophorus, the causative genetic information for melanoma formation has been assigned by classical genetics to chromosomal loci, which are located on the sex chromosomes. In our attempts to molecularly clone these melanoma-determining loci, named Tu, we have looked for restrictionfragment-length markers (RFLMs) linked to the Tu loci. These RFLMs should be useful in obtaining a physical map of a Tu locus, which will aid in the cloning of the corresponding sequences. DNA samples from various Xiphophorus strains and hybrids including those bearing different Tu wildtype, deletion and translocation chromosomes, were screened for the presence of random RFLMs using homologous or heterologous sequences as hybridization probes. We find an EcoRI restriction fragment which shows limited crosshybridization to the v e r b B gene-but not representing the authentic c-erb B gene of Xiphophorus-to be polymorphic with respect to different sex chromosomes. Linkage analysis revealed that a 5-kb fragment is linked to the Tu-Sd locus on the X chromosome, a 7-kb fragment is linked to the Tu-Sr locus on the Y chromosome, both of Xiphophorus macuhtw, and that a 12-kb fragment is linked to the Tu-Li locus on the X chromosome of Xiphophom variatw. Using different chromosomal mutants this RFLM has been mapped to a frequent deletion/ translocation breakpoint of the X chromosome, less than 0.3 cM apart from the Tu locus. I N Xiphophorus, certain hybrid genotypes develop spontaneous malignant melanoma. Melanoma formation has been attributed by classical genetic findings to the overexpression of a cellular gene, termed Tu. In nontumurous fish Tu was proposed to be negatively controlled by cellular egulatory genes (more recently termed antioncogenes by some authors) (for review, see ANDERS et al. 1984). In a typical crossing experiment a female Xiphophorms maculatw (platyfish) containing a specific Tu locus and its corresponding regulatory gene, which are both located on different chromosomes, is crossed with a male Xiphophorms helleri (swordtail), which is thought not to contain this particular Tu locus and its corresponding regulatory gene. Backcrossing of the Tucontaining hybrids to X . helleri results, in effect, in the progressive replacement of regulatory gene bearing chromosomes originating from the X . maculatw by chromosomes of X . helleri. This stepwise elimination of regulatory genes allows increased expression of Tu, resulting in the development of malignant melanoma in the hybrids. A lot of the information on the genetic basis for melanoma formation was gained thanks to the fact that in most cases the melanoma-determining locus Tu, besides often being linked to a pterinophore locus, is genetically associated with a locus for melanophore pigmentation patterns. These patterns are formed by a type of very large pigment cells resemGenetics 119: 679-685 (July, 1988). bling those cells which also were found in the melanoma of the tumorous hybrids. This pigment cell type has been referred to as macromelanophore (GORDON 1958) or more recently “transformed” (tr)melanophore (ANDERS et al. 1979). Various tr-melanophore patterns, differing with respect to the region of the body surface where the pigment cells are located, have been found in the wild-type populations of Xiphophorus. The different patterns are determined by dominant acting genetic loci, the majority of which are located on the sex-determining chromosomes (see KALLMAN 1975). All of them harbor the melanoma-determining Tu locus (ANDERS et al. 1984). The molecular nature of the color genes, the oncogene Tu and its corresponding regulatory genes is unknown. T o obtain information on the molecular genetics of melanoma formation, cloning of the melanoma-determining loci is required. So far, any approach in the Xiphophorus system to analyze the molecular genetics of the regulation of tumor expression failed due to the lack of molecular markers for the loci involved in oncogenesis. Our approach is based on (1) the availability of several mutants in Xiphophorus affecting the process of tumor formation as well as the wild-type pigmentation patterns; (2) a DNA transfection bioassay for Tu (for details on the assay, see VIELKIND et al. 1982); and (3) subtraction-cloning strategies utilizing Tu-wild-type